Predictable CRISPR/Cas9-Mediated COL7A1 Reframing for Dystrophic Epidermolysis Bullosa

Abstract End joining-based gene editing is frequently used for efficient reframing and knock-out of target genes. However, the associated random, unpredictable and often heterogeneous repair outcomes limit its applicability for therapeutic approaches. Recent studies revealed more precise and predictable outcomes simply based upon the sequence context at the CRISPR/Cas9 target site. The severe dystrophic form of the blistering skin disease epidermolysis bullosa (DEB) represents a suitable model platform to test these recent developments for the disruption and reframing of dominant and recessive alleles, respectively, both frequent in DEB. We delivered a CRISPR/Cas9 nuclease as ribonucleoprotein (RNP) into primary wild-type and recessive DEB (RDEB) keratinocytes to introduce a precise predictable single adenine sense-strand insertion at the target site. We achieved C7 knock-out in >40% of RNP-treated primary wild-type keratinocytes and C7 restoration in >70% of RNP-treated RDEB keratinocytes. Next generation sequencing of the on-target site revealed the presence of the precise adenine insertion upstream of the pathogenic mutation in at least 17% of all analyzed COL7A1 alleles. This demonstrates that COL7A1 editing based on precise end joining-mediated DNA repair is an efficient strategy to revert the disease-associated nature of DEB, regardless of the mutational inheritance.